Method and process to make and use cotton-tipped electrochemical immunosensor for the detection of corona virus

ABSTRACT

A method and process to make and use cotton-tipped electrochemical immunosensor for the detection of corona viruses is described. The immunosensor were fabricated by immobilizing the virus antigens on carbon nanofiber-modified screen printed electrodes which were functionalized by diazonium electrografting and activated by EDC/NHS chemistry. The detection of virus antigens were achieved via swabbing followed by competitive assay using fixed amount of antibody in the solution. Ferro/ferricyanide redox probe was used for the detection using square wave voltammetric technique. The limits of detection for our electrochemical biosensors were 0.8 and 0.09 pg/ml for SARS-CoV-2 and MERS-CoV, respectively indicating very good sensitivity for the sensors. Both biosensors did not show significant cross reactivity with other virus antigens such as influenza A and HCoV, indicating the high selectivity of the method.

FILED OF TECHNOLOGY

A method and process to make and use cotton-tipped electrochemicalimmunosensor for the detection of corona viruses is described.

BACKGROUND

The newly identified severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is the last discovered member of the corona viruses thatcause serious human respiratory infections. Other types of coronaviruses were previously known such as the Middle East respiratorysyndrome coronavirus (MERS-CoV), SARS-CoV1, HCoV-OC43, HCoV-229E, HCoVHKU1 and HCoV NL63. Since its first identification in China in 2019until present, SARS-CoV-2 has spread globally causing significantmorbidity and mortality. COVID-19; the disease caused by SARS-CoV-2; wasdeclared as pandemic by the world health organization on March 2020.Until now, there are no available vaccines or drugs proven to treatCOVID 19. Therefore, the timely detection of SARS-CoV-2, is urgentlyneeded to effectively control the rapid spread of the infection.

The testing of the virus can be achieved by reverse transcriptionpolymerase chain reaction (RT-PCR) test, detection of antigens, or byserological testing (the detection of the virus antibody). However, theserological tests are not reliable for the early diagnosis of SARS-CoV-2infection due to the relatively long delay between infection andseroconversion. Molecular diagnosis using RT-PCR is the primary usedmethod for the detection of corona viruses. However, PCR takesrelatively long time for analysis (minimum of 3 hours), and requiresseveral steps including the collection of the specimens by swabbing, thetransport of the sample into a solution and extraction of the viral RNAbefore amplification. Moreover, RT-PCR is relatively expensive whichhindered its wide applicability for population scale diagnosis ofSARS-CoV-2, particularly in low and middle income countries. Thus,sensitive, rapid and accurate diagnostic methods based on the directdetection of the viral antigens without pretreatment is highly demandedto control the COVID 19 outbreak. There are four main structuralantigens for corona viruses: nucleocapsid (N), spike (S), matrix (M),and envelope (E). Among them, the S and N proteins have the potential tobe used as biomarkers because they can distinguish different types ofcorona viruses.

Several diagnostic methods are being developed for the detection ofCOVID 19. Biosensors have been widely used for many diagnosticapplications showing fast, easy and reliable detection. Until now, onlyfew biosensors have been developed for SARS-CoV-2 such as thegraphene-based field-effect transistor (FET) biosensor reported by Seo.et al. The FET immunosensor was used for the detection of SARS-CoV-2using spike 51 protein as biomarker. Plasmonic photothermal biosensorsfor SARS-CoV-2 through nucleic acid hybridization have been alsodeveloped. Half-strip lateral flow assays (LFA) for the detection of Nprotein was reported. However, LFA provide qualitative orsemi-quantitative results and more work is still required to developmore accurate detection methods.

Electrochemical biosensors are one of the most popular types ofbiosensors which offer several advantages such as the low cost,capability of miniaturization, high sensitivity and selectivity. Theseadvantages make them ideal for use as point-of-care devices fordiagnostic applications. Electrochemical biosensors have been widelyintegrated with carbon nanostructures to fabricate highly sensitivedevices. Carbon nanofiber (CNF) is one of the materials that showedexcellent applications in biosensors because of its large surface area,stability and ease of functionalization.

Cotton swabs have been recently used in the fabrication of immunoassaysfor the detection of different pathogens. In these assays, thecolorimetric detection was achieved based on visual discrimination ofthe color change. These assays are simple, fast and easy to perform.However, they only give qualitative or semi-quantitative results. Thus,more accurate methods are still required.

SUMMARY

In the instant disclosure, a novel method and process to make and usecotton-tipped electrochemical immunosensor for the detection of coronavirus is described. In one embodiment a rapid test for severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2) is described. In anotherembodiment, a process using a cotton-tipped electrochemical immunosensorusing square wave voltammetry for detecting corona virus is described.In another embodiment, a mammalian is tested by swabbing nasal cavity,oral cavity or any other bodily fluid and or mucous membrane isdescribed.

In one embodiment, a cotton-tipped electrochemical immunosensor for thedetection of corona virus antigens is described. In another embodiment,a process and method of integrating the sample collection and detectiontools into a single platform by coating screen printed electrodes withabsorbing cotton padding is described. The immunosensor was fabricatedby immobilizing the virus antigen on carbon nanofiber-modified screenprinted electrodes which were functionalized by diazoniumelectrografting and activated by EDC/NHS chemistry. The detection ofvirus antigens were achieved via swabbing followed by competitive assayusing fixed amount of antibody in the solution. Ferro/ferricyanide redoxprobe was used for the detection using square wave voltammetrictechnique. The limits of detection for our electrochemical biosensorswere 0.8 and 0.09 pg/ml for SARS-CoV-2 and MERS-CoV, respectivelyindicating very good sensitivity for the sensors. Both biosensors didnot show significant cross reactivity with other virus antigens such asinfluenza A and HCoV, indicating the high selectivity of the method. Inone embodiment, the biosensor was successfully applied for the detectingof the virus antigens in spiked nasal samples showing excellent recoverypercentages. In one embodiment, the electrochemical immunosensor is adiagnostic tool for the direct rapid detection of the corona virusesthat requires no sample transfer or pretreatment. In another embodiment,cotton-tipped electrochemical immunosensor plays dual function roles assample collector as well as detector allowing the rapid, simple and lowcost detection of the viruses without prior sample preparation.

CNF-modified screen printed carbon electrodes were used for theimmunosensor fabrication on which the S or N antigens were immobilizedafter functionalization of the sensor surface by electrografting.Competitive assay was used for the detection of the S and N proteinsshowing excellent sensitivity and selectivity.

In one embodiment, a process of diagnosing a viral infection isperformed using the steps of modifying an electrode using an electrodematerial to make a modified electrode; functionalizing the modifiedelectrode by grafting a carboxy phenyl group to make a functionalelectrode; immobilizing a viral antigen on the functional electrode tomake a viral antigen coated functional electrode; capping the viralantigen coated functional electrode with an layer of tip material tomake a tipped electrochemical sensor; contacting the tippedelectrochemical sensor with a mammalian body part which releases amucous membrane secretion on the tipped electrochemical sensor;immersing the sample collected by the tipped electrochemical sensor intoa tube containing a redox solution; and applying a voltage difference onthe sample collected on the tipped electrochemical sensor in the tubecontaining redox to read a difference in a reduction peak current orcharge transfer resistance using a square wave voltammetry orelectrochemical impedance spectroscopy.

In another embodiment a method of diagnosing a viral infection is doneby the following steps of modifying an electrode using carbon nanofiberto make a modified carbon electrode; grafting a carboxy phenyl group tomake a functional carbon electrode by functionalizing the modifiedcarbon electrode; coating a viral antigen on the functional carbonelectrode to make a coated functional electrode; layering a cotton layerto make a cotton tipped electrochemical sensor on the viral antigencoated functional electrode with; collecting a sample from a mammalianbody part infected by a virus which releases a mucous membrane secretionon the cotton tipped electrochemical sensor; immersing the samplecollected cotton tipped electrochemical sensor into a tube containing aredox solution; applying a voltage difference on the sample collectedcotton tipped electrochemical sensor in the tube containing redox toread a difference in a reduction peak current; and identifying thepresence or absence of viral infection based on reduction peak currentdifference.

Other features will be apparent from the accompanying drawings and fromthe detailed description that follows.

BRIEF DESCRIPTION OF DRAWINGS

Example embodiments are illustrated by way of example and not limitationin the figures of the accompanying drawings, in which like referencesindicate similar elements and in which:

FIGS. 1A, 1B and 1C show steps of sensor fabrication, sample collectionand competitive detection of corona virus.

FIG. 2 shows a flow chart of the process and method of making and usingthe cotton tipped electrochemical sensor.

FIGS. 3A, 3B, 3C and 3D show scanning microscope images of the carbonscreen printed electrodes and the carbon electrode after modificationwith carbon fibers and the The X-ray photoelectron spectroscopy C1s highresolution spectra of the modified electrodes before and after electrografting.

FIG. 4 shows the Square wave voltammograms of different electrodes.

FIGS. 5A, 5B, 5C, 5D, 5E and 5F shows the various steps of using thecotton tipped electrochemical sensor.

FIGS. 6A, 6B, 6C and 6D shows square wave voltammograms and detectioncurves of the MERS-CoV (A) and SARS-CoV-2 (C) immunosensors before andafter binding with different concentrations of spike 51 and N antigens.

FIGS. 7A and 7B shows the MERS-CoV and SARS-CoV-2 biosensors responsetowards the binding with Spike 51, N, Flu A and HCoV antigens.

Other features of the present embodiments will be apparent from theaccompanying drawings and from the detailed description that follows.

DETAILED DISCUSSION

The instant disclosure describes a process and method for a rapid testfor infectious disease such as COVID-19. This particular pandemic andother pandemics that spread and infect mammalians in a short duration.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)pandemic has led to an urgent need for low-cost and rapid diagnostictools to enable the infection control and suppress the spread of theCOVID 19 disease. The shorter the process and simpler the method it iseasier for public health authorities to implement it. The instantlydisclosed process and method for electrochemical immunosensor is apromising diagnostic tool for the direct rapid detection of the coronaviruses that requires no sample transfer or pretreatment.

Our novel process and method is the combination of cotton fiber andelectrochemical assay for the detection of SARS-CoV-2 and MERS-COVprotein antigens. The cotton-tipped electrochemical immunosensor playsdual function roles as sample collector as well as detector allowing therapid, simple and low cost detection of the viruses without prior samplepreparation.

Materials and reagents: Potassium ferricyanide (K₃Fe(CN)₆), potassiumferrocyanide (K₄Fe(CN)₆), 4-aminobenzoic acid, hydrochloric acid, sodiumnitrite, bovin serum albumin (BSA) and phosphate buffer saline (PBS)were obtained from Sigma (Ontario, Canada)(http://www.sigmaaldrich.com/canada-english.html). N-hydroxysuccinimide(NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) and PCR tubes were purchased from Fisher Scientific (Ontario,Canada). Carbon nanoFibers powder was obtained from Metrohm DropSens,Inc. (Asturias, Spain). Antigen of MERS-CoV (725 Spike protein 51) andthe antigen of SARS-CoV-2 (Nucleocapsid protein (N protein)) and theirantibodies were purchased from Sino Biological (Beijing, china)(https://www.sinobiological.com/). Influenza A antigen (N1H1) No. J8034were purchased from biospacific (CA, USA)(https://www.biospacific.com/). HCoV antigen (HK41 N) and its antibodywere obtained from Medix Biochemica (Finland). Sterile cotton wasobtained from local pharmacy in Riyadh city. 1×PBS buffer, pH 5.5 wasused for the preparation of EDC/NHS solution for the activation step.1×PBS buffer, pH 7.4 was used for the preparation of the antigens andantibodies solutions and washing steps. The CNF solution was prepared bydispersion of 1 mg of the CNF powder in one ml of DMF with sonicationfor 30 min until obtaining a homogeneous solution. All the solutionswere prepared using Milli-Q water.

Instrumentation: An Autolab potentiostat, PGSTAT302N from (Metrohm,Switzerland) was used to perform all the electrochemical measurements(the cyclic and square wave voltammetry). Disposable screen printedelectrodes (PCR P01) adopted for PCR tubes were purchased from BioDeviceTechnology (Nomi, Japan). Each electrode consists of rectangle-shapedcarbon working and counter electrodes and a central silver/silverchloride (Ag/AgCl) reference electrode. The electrical contacts are madeof silver. The ends of the electrodes were designed to fit into thestandard PCR tubes. The electrodes were connected to the potentiostatthrough a connector obtained from BioDevice Technology. The morphologyof the CNF modified electrodes were examined via Scanning electronmicroscopy (SEM) measurements using an acceleration voltage of 5 kV,magnification=12000× and a working distance of 9.8 mm.

Modification of the carbon electrodes with carbon nanofibers: Dropcasting method was used to modify the carbon working electrode of thescreen printed chip. 0.5 μL of the CNF solution in DMF (1 mg/ml) wasplaced on the surface of the working electrodes. The electrodes werethen left to dry at room temperature for at least 20 hours. Then, theelectrodes were gently washed with water to remove the excess CNF anddried.

Method and process of functionalization of the carbon nanofiber-modifiedelectrodes using electrografting: The CNF surface was thenfunctionalized using electrografting of carboxyphenyl groups via thereduction of diazonium salt as previously reported on different carbonmaterials. Briefly, as shown in FIGS. 1A, 1B and 1C, 2 mM of4-aminobenzoic acid solution (102) were mixed with 2 mM of sodiumnitrite solution in 0.5 M HCl (103) with stirring for 10 min at roomtemperature to produce the diazonium salt (104). 100 μl of the diazoniumsolution was then added into a PCR tube in which the CNF electrode wasimmersed from one end and the other was connected to the potentiostat toperform electroreduction (105) using 2 cyclic voltammetry scans from+0.2 to −0.7 V at a scan rate of 50 mV s-1 to make the chips. The chipswere then washed with water and dried. To confirm the success of thegrafting step of the carboxyphenyl groups on the electrode surface,X-ray photoelectron spectroscopy (XPS) measurements were recorded forthe CNF-modified electrodes before and after the electroreduction step.

Immobilization of the virus antigens on the functionalized electrodesand preparation of the cotton-based electrochemical sensor was carriedout in step 204 as also shown in FIG. 2. The carboxyphenyl-modified CNFelectrodes (106) were incubated in PBS buffer, pH 5.5 containing 100 mMEDC and 20 mM NHS (107) for 1 hour at room temperature in order toactivate the terminal carboxylic groups. After that, the electrodes werewashed with PBS buffer pH 7.4 and incubated individually with 10 μg/mlof either the MERS-CoV or SARS CoV-2 antigens solutions (107) in PBSbuffer, pH 7.4 for 3 hours in a water-saturated atmosphere at roomtemperature. Finally, the antigen-coated electrodes (108) were thenrinsed using PBS buffer and incubated in a solution of 0.1% BSA in PBSbuffer pH 7.4 for 30 min to block the free sites on the electrodesurface.

After immobilizing the antigens, the cotton-tipped immunosensor (109)were prepared by covering the tapering end of the electrode containingthe detection zone with a piece of cotton Fiber (30 mg) withoutscratching the sensor surface (FIG. 2, step 206). The preparedimmunosensor can be used immediately for collecting the nasal sample(110) (FIG. 1B) (FIG. 2, step 208) or kept dry at 4° C. until furtheruse (108).

Electrochemical competitive detection of MERS-CoV and SARS-CoV-2 on thecotton-based immunosensor: For the detection of the standard antigenssolutions in PBS buffer, different concentrations (0.1 pg/ml to 1 μg/ml)of each antigen (the spike S1 or the N protein) was firstly mixed with10 μg·mL⁻¹ solution of the respective antibody in PBS buffer pH 7.4 offthe chip in an Eppendorf Tube (112). Then, 100 μl of each mixture wasadded to a PCR tube in which the cotton-tipped immunosensor was thenimmersed and incubated for 20 min at room temperature (111). After theincubation, the immunosensor was taken out and placed in another PCRtube containing 100 μl PBS buffer and left for 1 min for washing. Then,the cotton electrode was placed on absorbing cotton pad to remove theexcess washing solution. Finally, the immunosensor was immersed into aPCR tube containing 10 mM of the redox solution (ferro/ferricyanide) inPBS buffer pH 7.4 to maintain all electrodes in contact with thesolution during the SWV measurements (FIG. 2, step 210). Theelectrochemical sensor response was calculated as (i−i^(o))/i^(o)%where, i is the reduction peak current of the electrodes afterincubation with the mixture of the antigen and antibody solution andi^(o) is the original peak current of the immunosensor beforeincubation.

Electrochemical measurements: The Square wave voltammetry measurements(SWV) were recorded in 10 mM 1:1 ferro/ferricyanide solution in 10 mMPBS buffer pH 7.4 (FIG. 2, step 212). The scanning potential range ofthe SWV is from 0.3 to −0.5 V at step potential of −5 mV, amplitude of20 mV and frequency of 25 Hz. Base-line corrections were performed forall the SWV curves. The cyclic voltammetry measurements (C) for thediazonium electroreduction was performed at a scan rate of 50 mV s-1 byCV scanning from +0.2 to −0.7 V (113).

Cross reactivity testing of the immunosensor with other antigens: Inorder to study the selectivity of the MERS-CoV and SARS-CoV-2immunosensor towards their antigens, the immunosensor were testedagainst Flu A and HCoV antigens. The test was performed by mixing 10 μlof MERS-CoV antibody (10 μg/ml) with 10 μl of Flu A or HCoV antigensolution (1 ng/ml) and 10 μl of SARS-CoV-2 antibody (10 μg/ml) with 10μl of Flu A or HCoV antigens (1 ng/ml). Then, the mixtures wereincubated with each immunosensor for 20 min and then washed. The sensorresponse in each case was evaluated as described above.

Testing of the immunosensor on spiked nasal fluid: The cotton-tippedimmunosensor were used to collect nasal fluid from healthy volunteer.Then the immunosensor was immersed in PCR tube containing 50 μl of 10μg/ml solution of spike S1 antibody spiked with 50 μl of 0.001 or 100ng/ml of the spike S1 protein solution in PBS buffer pH 7.4. Theimmunosensor were incubated for 20 min then washed and measured by SWVas described above.

The method and process is described in FIG. 2. The start 200 ismodification of the electrode using carbon nanofiber (CNF) (201). Theentire process and method is done to create cotton tippedelectrochemical sensor, method of using it to collect sample and finallymeasuring the results using square wave voltammetry and reading themeasurements to detect the said virus. The electrode material can be oneof a carbon nanofiber, carbon nanotubes, graphene or any other carbonnanomaterial. And the tip/cover material can be cotton or othersynthetic fiber such as nylon, Rayon, polyurethane foam, and Polyester.And the electrochemical measurements can be done using square wavevoltammetry or electrochemical impedance spectroscopy.

A process to functionalize the modified carbon electrode is done bygrafting carboxyphenyl groups to the surface (202). FIG. 1 A describesthis method and process in detail. Immobilization of viral antigen onthe functionalized electrode is described in FIG. 1A step 109. Step 206describes capping the antigen immobilized functionalized electrode withcotton layer to create cotton tipped electrochemical sensor or any otherlayer that can absorb the sample and yet be electro sensitive isimportant.

Characterization of the carbon nanofiber-modified electrodes before andafter the electrografting of carboxyphenyl moieties: In this work, weused screen printed carbon electrodes for the sensor fabrication.Commercially available CNF powder in DMF was used to modify the carbonworking electrodes. Scanning electron microscopy (SEM) was used tocharacterize the morphology of the carbon working electrodes as well asthe CNF-modified electrodes. As shown in FIGS. 3A and 3B, the SEM imagesof the carbon and CNF-modified electrodes exhibited differentmorphologies. The carbon electrode showed a typical multilayeredgraphitic structure, whereas, the CNF electrodes exhibited a denselypacked layer of CNF rods indicating higher surface area of the CNFelectrodes compared with the unmodified carbon electrode.

The CNF electrodes were then functionalized by electrografting of thecarboxyphenyl layer in the electrode surface. The electrografting wasachieved via electroreduction of carboxyphenyl diazonium salt by CV. Asshown in FIG. 3C, the first CV scan showed a single irreversiblecathodic peak at 0.3 V, characteristic for the reduction of diazoniumsalt via one electron transfer process which led to the removal ofnitrogen molecule and the formation of aryl radical that form a covalentbond with the CNF surface. However, in the second CV scan, the reductionpeak was almost disappeared likely due to the complete coverage of theCNF electrode surface with the carboxyphenyl layer which retarded theelectron transfer process. Further CV scans will lead to a buildup ofmultilayers which will negatively impact the biosensor performance aspreviously reported. Thus, only 2 CV scans were performed for theelectrografting step to ensure monolayer formation of the carboxyphenylmoieties.

XPS was then used to analyze the CNF electrode surface before and afterthe electrografting step using CV. FIG. 3D shows the XPS C1s highresolution spectra of the electrode before and after functionalization.A new peak was observed clearly at 288.8 eV after the electrograftingstep compared with the spectra of the unmodified CNF electrode whichconfirms the successful attachment of the carboxyl groups on the CNFsurface.

Square wave voltammetry characterization of the stepwise fabricationprocess of the biosensor: To characterize the fabrication steps of theimmunosensor, SWVs were recorded at different steps in ferro/ferricyandesolution. FIG. 4 shows the SWV reduction peaks of the redox couple atthe bare carbon electrode, the CNF-modified electrode, thecarboxyphenyl-modified electrode and after the immobilization of thespike S1 protein. As shown in the figure, an enhancement of thereduction peak current was observed when the electrode was modified withCNF compared to the bare carbon electrode. This is attributed to theincrease in the electrochemical surface area of the electrode because ofthe CNF material. However, after the electroreduction step of thediazonium salt on the CNF electrode, the SWV reduction signal of theredox couple was almost disappeared due to the formation of thecarboxyphenyl layer on the electrode surface. This has led to thepassivation of the electrode and retardation of the electron transferbecause of the aromatic layer. Moreover, the negatively charged carboxylgroups repelled the redox anion from the surface leading to a decreasein the reduction current. However, the immobilization of the virusprotein antigen on the carboxyphenyl-modified electrode after activationwith EDC/NHS led to an increase in the reduction peak current. This islikely attributed to the shielding of the negatively charged carboxylicgroups on the CNF surface with the antigens.

Effect of the cotton coating on the electrochemical signal of theelectrode: The goal of this work is to develop a cotton-tippedelectrochemical immunosensor which can perform both the samplecollection as well as the detection. After the immobilization of theprotein antigen on the CNF electrode, the electrodes were blocked withBSA solution and left to dry at room temperature. Then, a piece ofcotton Fiber was then coated on the detection zone of the electrode asshown in FIG. 5A which mimics the standard Q-Tip. The cotton was usedbecause of its high absorbing capability which allows the collection ofthe nasal samples by swabbing. The incubation of the sensor with theantibody solution as well as the measuring redox solution is done in PCRtubes as shown in FIGS. 5B and 5C.

Investigation was carried out to find out if the coating of theelectrode with the cotton fiber impacts the electrochemical signal. FIG.5D shows the SWV reduction peak current of the bare electrode before andafter coating with the cotton in the redox solution. Non-significantchange (less than 5%) in the electrochemical signal was observed aftercoating the carbon electrode with the cotton likely due to the highabsorbing properties of the cotton Fiber. The cotton fiber was able toabsorb the redox solution by capillary action and transports it to theelectrode surface which remains in contact with the solution during themeasurements. FIG. 5E shows the reduction peak current at MERS-CoVimmunosensor (the spike S1 modified electrode after blocking with BSA)before and after coating with the cotton. The change in the peak currentwas less than 5% indicating that there was no significant effect on theelectrochemical signal of the sensor due to the coating with the cotton.It was also important to assess the effect of cotton coating on thebiosensor response due to the binding of the antigen on the sensorsurface with the antibody in the solution. For this purpose, thereduction current of the uncoated and cotton coated immunosensor weredetected in the redox solution. Then the two immunosensor were incubatedin 1 ng/ml solution of antibody in two PCR tubes. After washing, theelectrodes were measured in the redox solution and the sensor responsein was evaluated as the percentage change in the reduction peak currentin each case. As shown in FIG. 5F, the cotton-tipped biosensor responsewas almost similar to the uncoated immunosensor. These results implythat the cotton-tipped electrochemical sensor can be used as aneffective platform with dual function as a sample collector anddetection tool.

Competitive electrochemical detection of MERS-CoV and SARS-CoV-2 on theimmunosensor: The detection on the immunosensor was achieved viacompetitive assay where a fixed concentration of antibody is mixed inthe solution with different concentrations (0.1 pg/ml to 1 μg/ml) of thespike S1 or N protein antigen solution and then incubated on theimmunosensor surface. A competition between the immobilized and the freeantigen to bind the free antibody in the solution is realized. Thehigher the concentration of free antigen, the smaller the amount ofantibody available to bind to the antigen on the electrode surface. Thebinding of the antibody to the antigen on the immunosensor causes anincrease in the reduction peak current of the ferro/ferricyanide redoxcouple. This could be attributed to the binding with the positivelycharged antibodies which attract the redox anions leading to anenhancement of the reduction current.

FIG. 6A shows the SWVs of the MERS-CoV immunosensor upon incubation withdifferent concentrations of spike S1 protein mixed with 10 μg/ml of theantibody solution. When the concentration of the spike S1 protein washigh, the increase in the SWV peak current was low and vice versa.Similarly, the SWVs of the SARS-CoV-2 immunosensor upon incubation withdifferent concentrations of nucleocapsid protein mixed with 10 μg/ml ofthe antibody solution are shown in FIG. 6C. The calibration plots of theMERS-CoV and SARS-CoV-2 immunosensor are shown in FIGS. 6B and 6D,respectively. The calibration plot is obtained by plotting the biosensorresponse (the percentage increase in the peak current; (i−i^(o))/i^(o)%)versus the logarithm of the antigen concentration. Good linearrelationship was obtained for the concentration ranges from 0.1 pg/ml to1000 ng·mL-1 and 1 to 1000 ng·mL-1 for MERS-CoV and SARS-CoV-2,respectively. The linear regression equations of the two straight lineswere: (i−io)/io %=477.8+−124.6 log C (ng/ml), R=0.992 for MERS-CoV and(i−i^(o))/i^(o)%=63.6+−7.8 log C (ng/ml), R=0.991 for SARS-CoV-2. Thelimits of detection (LODs) were calculated to be 0.8 and 0.09 pg/ml forSARS-CoV-2 and MERS-CoV, respectively indicating excellent sensitivityof the immunosensor. These LODs are much lower than other reportedimmunoassays such as ELISA (LOD of ELISA is 0.4 ng/ml and 1 ng/ml forSARS-CoV-2 and MERS-CoV, respectively). All the experiments were done intriplicates and the standards deviations of the measurements wereranging from 2.5 to 5.5% indicating excellent reproducibility of the twoelectrochemical immunosensor.

Cross reactivity of the MERS-CoV and SARS-CoV immunosensor with othervirus antigens: In order to confirm the selectivity of our immunosensorto the Spike S1 and nucleocapsid proteins, the immunosensor were testedagainst other virus antigens such as Flu A and HCoV. FIGS. 7A and 7Bshow the MERS-CoV and SARS-CoV immunosensor responses against Spike S1and nucleocapsid proteins as well as HCoV and Flu A. As shown in thefigure, significant difference between the response of each immunosensortowards its specific and nonspecific antigens. Higher sensor responsewas obtained in the case of the nonspecific antigens because there wereno binding in the solution and thus, the maximum amount of antibody wasfree to bind to the electrode whereas, lower response was obtained whenthe specific antigen was used. These results indicate high selectivityof the MERS-CoV and SARS-CoV electrochemical immunosensor.

Application of the cotton-tipped immunosensor in spiked nasal samples:To investigate the practical applicability of the developedcotton-tipped electrochemical immunosensor for the detection of thevirus in the nasal fluid, the sensor was used to collect the nasal fluidfrom healthy volunteer and then subjected to the electrochemicalmeasurements as described in the experimental section. Table 1 showsvery good recovery percentages (91 to 95.5%) of the spike S1 protein onthe MERS CoV cotton immunosensor. This indicates the success of thecotton immunosensor to collect as well as detect the virus protein withhigh accuracy and without significant interference from the othercomponent of the nasal fluid.

TABLE 1 the real sample application of the MERS-CoV immunosensor inspiked nasal samples (n = 3) showing the recovery percentages. SpikedMERS- COV ng/mL Recovery % RSD % 0.001 95.5 5.2 100 91 6.0

What is claimed is:
 1. A process to diagnose a viral infection,comprising: modifying an electrode using an electrode material to make amodified electrode; functionalizing the modified electrode by grafting acarboxy phenyl group to make a functional electrode; immobilizing aviral antigen on the functional electrode to make a viral antigen coatedfunctional electrode; capping the viral antigen coated functionalelectrode with an layer of tip material to make a tipped electrochemicalsensor; wherein the layer of tip material is one of a cotton, nylon,rayon, polyurethane foam or polyester; contacting the tippedelectrochemical sensor with a mammalian body part which releases amucous membrane secretion on the tipped electrochemical sensor;immersing the sample collected by the tipped electrochemical sensor intoa tube containing antibody solution and a redox solution; and applying avoltage difference on the sample collected on the tipped electrochemicalsensor in the tube containing redox to read a difference in a reductionpeak current using a square wave voltammetry or a charge transferresistance using electrochemical impedance spectroscopy usingSmartphone.
 2. The process of claim 1, wherein the reduction peakcurrent is done using the square wave voltammetry.
 3. The process ofclaim 1, wherein the charge transfer resistance is done using theelectrochemical impedance spectroscopy.
 4. The process of claim 1,wherein the mammalian body part is one of a nasal cavity, oral cavity orcorneal surface.
 5. The process of claim 1, wherein the layer of tipmaterial is a cotton.
 6. The process of claim 1, wherein the electrodematerial is one of a carbon nanofiber, carbon nanotubes, graphene or anyother carbon nanomaterial.
 7. The process of claim 6, wherein theelectrode material is the carbon nanofiber.
 8. The process of claim 1,wherein the viral infection is due to a Corona Virus.
 9. A process todiagnose a viral infection, comprising: modifying an electrode usingcarbon nanofiber to make a modified carbon electrode; functionalizingthe modified carbon electrode by grafting a carboxy phenyl group to makea functional carbon electrode; immobilizing a viral antigen on thefunctional carbon electrode to make a viral antigen coated functionalelectrode; capping the viral antigen coated functional electrode with acotton layer to make a cotton tipped electrochemical sensor; contactingthe cotton tipped electrochemical sensor to a mammalian body part whichreleases a mucous membrane secretion on the cotton tippedelectrochemical sensor; immersing the sample collected cotton tippedelectrochemical sensor into a tube containing antibody and a redoxsolution; and applying a voltage difference on the sample collectedcotton tipped electrochemical sensor in the tube containing redox toread a difference in a reduction peak current.
 10. The process of claim1, wherein the viral infection is due to a Corona Virus.
 11. A method ofdiagnosing a viral infection, comprising; modifying an electrode usingcarbon nanofiber to make a modified carbon electrode; grafting a carboxyphenyl group to make a functional carbon electrode by functionalizingthe modified carbon electrode; coating a viral antigen on the functionalcarbon electrode to make a coated functional electrode; layering acotton layer to make a cotton tipped electrochemical sensor on the viralantigen coated functional electrode with; collecting a sample from amammalian body part infected by a virus which releases a mucous membranesecretion on the cotton tipped electrochemical sensor; immersing thesample collected cotton tipped electrochemical sensor into a tubecontaining antibody and redox solution; applying a voltage difference onthe sample collected on the cotton tipped electrochemical sensor in thetube containing redox to read a difference in a reduction peak current;and identifying the presence or absence of viral infection based oncurrent difference.
 12. The method of claim 11, wherein the viralinfection is due to a Corona Virus.
 13. The method of claim 11, whereinthe reduction peak current is done using the square wave voltammetry.